The MutL homolog 1 (MLH1) gene is a tumor suppressor gene involved in the DNA mismatch repair pathway. Germline pathogenic variants in MLH1 can cause cancer-predisposing Lynch syndrome, with increased susceptibility to colorectal cancer in particular.

An important part of the screening and diagnosing strategy is the molecular analysis determining whether the MLH1 promoter is hypermethylated. The presence of MLH1 promoter hypermethylation is associated with sporadic cancers, while the absence of methylation indicates Lynch syndrome.

MLH1 is a part of the cellular DNA repair system and specifically plays a role in the DNA mismatch repair pathway. The MLH1 gene is located on chromosome 3p21. The genes involved in DNA mismatch repair are highly conserved throughout evolution.

In humans, they exist as homologs of the prokaryotic MutS and MutL proteins, known as MSH2, MSH6, MLH1, MLH3, PMS1, and PMS2. In eukaryotes, the DNA mismatch repair proteins exert their functions as hetero-dimers rather than monomers.

The MutS homologs exist in two forms, MutSα (MSH2-MSH6) and MutSβ (MSH2-MSH3). MutSα handles single-base mismatches and small insertion/deletion (indel) mismatches, while MutSβ repairs larger indel mismatches. The MutL homologs exist as three heterodimers, MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (MLH1-MLH3). MutLα is the most active complex and supports repair initiated by the MutS complex.

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MLH1 gene in DNA mismatch repair

DNA mismatch repair is active primarily during DNA replication and recombination. In DNA mismatch repair, mismatch lesions are recognized by the MutS complex. The localization of MutS to the DNA lesion recruits the MutL complex to the site.

MutL possesses endonuclease activity and nicks the DNA backbone near the lesion. The rest of the repair can then proceed through either an EXO1-dependent or EXO1-independent pathway. In the EXO1-dependent pathway, the MutS-MutL complex recruits the exonuclease Exo1 for the excision of the lesion in coordination with Proliferating Cell Nuclear Antigen (PCNA).

The single-stranded DNA intermediate is stabilized by Replicating Protein A (RPA). DNA polymerase δ then fills in the gap with newly synthesized DNA. In the EXO1-independent pathway, DNA polymerase δ-mediated DNA synthesis displaces one of the DNA strands, resulting in the formation of a 5′-flap.

This flap is then removed by FEN-1. In both cases, DNA ligase seals the resulting nick in the DNA backbone.

Germline mutations in the DNA mismatch repair genes result in the accumulation of mutations during DNA replication, especially in the repetitive DNA sequences known as microsatellites.

The DNA mismatch repair deficiency gives rise to variations within the microsatellites, manifesting as loss or gain in repeat length. This is known as microsatellite instability (MSI).

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Colorectal cancer

Colorectal cancer (CRC) is one of the most common cancers and causes of cancer-related deaths globally. Most cases are in Western countries, with an increasing incidence.

About 70% of all CRC cases are sporadic, while familial CRC accounts for ~25%, and inherited CRC is around 5%. Familial CRC is defined based on family history, and inherited CRC is due to germline mutations that follow Knudson’s 2-hit model. There are three underlying pathogenic mechanisms leading to CRC: Chromosomal instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP).

The CIN pathway is also called the classical pathway, as it represents the majority of CRC cases.

An initiating mutation occurs in the adenomatous polyposis gene (APC) and triggers the formation of non-malignant polyps. The APC mutation is then followed by mutations in KRAS, TP53, and DCC. 

The MSI pathway leads to a hypermutated phenotype, which promotes the accumulation of mutations, leading to cancer when tumor suppressor genes or oncogenes are affected. 

The CIMP pathway leads to hypermethylation of tumor suppressor genes, resulting in gene silencing.

MLH1 gene in Lynch syndrome 

Approx. 5% of all CRCs are inherited. Lynch syndrome (previously known as hereditary nonpolyposis colorectal cancer, HNPCC) accounts for ~3% of CRCs and is caused by heterogenous germline mutations or (rarely) epimutations in one of the DNA mismatch repair genes MLH1, MSH2, MSH6, or PMS2, or by EPCAM deletion.

The majority of these mutations affect MLH1 or MSH2. Patients with this syndrome have an increased risk of not only CRC, but also endometrial cancer, ovarian cancer, stomach cancer, cancer of the urinary tract, and skin cancer. To identify patients with Lynch syndrome, all CRC cases are investigated for DNA mismatch repair deficiency.

It is also recommended that endometrial cancer cases are tested for DNA mismatch repair deficiency due to the relatively high frequency of endometrial cancer in Lynch syndrome.

For CRC, both inherited and sporadic cancers can be MSI-H. DNA mismatch repair deficiency is tested either by testing for MSI via PCR or for protein loss through immunohistochemistry. MSI-H tumors due to loss of MLH1 or PMS2, are additionally tested for MLH1 promoter hypermethylation and BRAF V600E mutation.

This is an important part of diagnosing, as it allows the distinction between sporadic and inherited cancer. If it is diagnosed as an inherited cancer, it can have consequences not only for the patient but also for their next of kin.

If MLH1 promoter hypermethylation or a BRAF V600E mutation is found, it suggests that the cancer is sporadic.

If there is a loss of DNA mismatch repair protein expression without MLH1 hypermethylation and BRAF V600E mutation, it indicates inherited cancer and the patient is referred to germline mutation testing.

DNA mismatch repair proficient tumors are thought to be sporadic and are associated with the CIMP phenotype.

Constitutional mismatch repair deficiency (CMMR-D) is a syndrome predisposing to cancer already during childhood and adolescence.

The cancers typical of this syndrome are hematological, of the brain, and colorectal, as well as other malignancies predisposed to by Lynch syndrome.

CMMR-D patients typically present with brain tumors and the mean age of diagnosis is 9 years old. The syndrome is caused by biallelic germline pathogenic variants in the DNA mismatch repair system.

When both parents have Lynch syndrome, there is a 25% risk of CMMR-D in the offspring. Although screening for DNA mismatch repair deficiency is not common in brain tumors, the identification of CMMR-D has important implications for the treatment of the patient and surveillance of both the patient and the patient’s family.

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How MethylDetect can assist you in your research

At MethylDetect, we can provide you with ready-to-use kits for DNA methylation analysis of your target of interest. In our catalog, we offer more than 850 EpiMelt assays.

In Products, you will find EpiMelt kits targeting genes relevant for colorectal cancer and DNA damage repair research, such as, but not limited to, MLH1, MSH2, MSH3, MSH6, PMS1, and PMS2.

The EpiMelt assay kits are based on the Methylation-Sensitive High-Resolution Melting (MS-HRM) technology and can be used with standard laboratory equipment for qPCR and melting assessment.

Each EpiMelt assay kit comes with a unique control system, securing high sensitivity. Please consult our catalog at Products, and the protocol at Assay Protocol MethylDetect, for further information on setting up the EpiMelt analysis in your laboratory.

Custom-Tailored EpiMelt Kits

If your target gene is not found in our portfolio, we offer to design and produce EpiMelt assay kits tailored to target specific areas of the genome.

Following methylation-specific array screening analyses, you may have identified targets, which are not yet described in the literature.

In collaboration with you, we can design and produce EpiMelt assay kits targeting these specific genomic areas, and tailor the kit to fulfill your needs.

We take into account if your samples are FFPE tissue, liquid biopsies, or high-quality DNA. Customer-tailored EpiMelt assays are always performed in close collaboration with you.

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Further reading

Calil, F. A., Li, B. Z., Torres, K. A., Nguyen, K., Bowen, N., Putnam, C. D., & Kolodner, R. D. (2021). Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae. Nat Commun, 12(1), 5568.

Idos, G., & Valle, L. (1993). Lynch Syndrome. In M. P. Adam, G. M. Mirzaa, R. A. Pagon, S. E. Wallace, L. J. H. Bean, K. W. Gripp, & A. Amemiya (Eds.), GeneReviews(®). University of Washington, Seattle. Copyright © 1993-2023, University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved.

Maratt, J. K., & Stoffel, E. (2022). Identification of Lynch Syndrome. Gastrointest Endosc Clin N Am, 32(1), 45-58.

Mármol, I., Sánchez-de-Diego, C., Pradilla Dieste, A., Cerrada, E., & Rodriguez Yoldi, M. J. (2017). Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer. Int J Mol Sci, 18(1).

Muro, Y., Sugiura, K., Mimori, T., & Akiyama, M. (2015). DNA mismatch repair enzymes: genetic defects and autoimmunity. Clin Chim Acta, 442, 102-109.

Onishi, S., Yamasaki, F., Kuraoka, K., Taguchi, A., Takayasu, T., Akagi, K., & Hinoi, T. (2023). Diagnostic and therapeutic challenges of glioblastoma as an initial malignancy of constitutional mismatch repair deficiency (CMMRD): two case reports and a literature review. BMC Med Genomics, 16(1), 6.